3680
J. Med. Chem. 2005, 48, 3680-3683
Letters Synthesis and Structure-Activity Relationship of 4-Substituted 2-(2-Acetyloxyethyl)-8-(morpholine4-sulfonyl)pyrrolo[3,4-c]quinoline1,3-diones as Potent Caspase-3 Inhibitors Dmitri V. Kravchenko,† Yulia A. Kuzovkova,† Volodymyr M. Kysil,‡ Sergey E. Tkachenko,‡,§ Sergey Maliarchouk,‡,# Ilya M. Okun,‡,# Konstantin V. Balakin,‡,§ and Alexandre V. Ivachtchenko*,‡ Departments of Organic Chemistry, Medicinal Chemistry, and Molecular Biology and HTS, Chemical Diversity Research Institute, Khimki, Moscow Reg., Russia, and ChemDiv, Inc., 11558 Sorrento Valley Road, Suite 5, San Diego, California 92121 Received December 14, 2004 Abstract: Synthesis, biological evaluation, and SAR dependencies for a series of novel 1,3-dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]quinoline inhibitors of caspase-3 are described. The inhibitory activity of the synthesized compounds is highly dependent on the nature of 4-substituents on the core scaffold. 4-Methyl- and 4-phenyl-substituted derivatives, which were the most active compounds within this series, inhibited caspase-3 with IC50 of 23 and 27 nM, respectively.
Caspases, a family of cysteine-dependent aspartatedirected proteases, comprise highly homologous enzymes that play an important role in apoptotic cell death.1 Caspase-3 (apopain) is situated at a key junction in the apoptosis, mediating apoptotic cascade from the intrinsic and extrinsic activation pathways.2 Therefore, caspase-3 is an attractive target for therapeutic intervention. For instance, inhibitors of caspase-3 were described as promising cardioprotectants,3 neuroprotectants,4 and antiarthritic agents.5 1,3-Dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]quinolines represent a relatively little explored class of heterocyclic structures with promising physiological activities. Thus, they were reported as cytotoxic agents6 and central nervous system active agents, selective agonists, antagonists, or inverse agonists for GABAA (γ-aminobutyric acid A) brain receptors.7 Recently, we described an efficient synthesis and biological activity of 8-sulfamide derivatives of 1,3-dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]quinolines.8 Synthesis of 5H-pyrrolo[3,4-c]quinoline-1,3,4-triones was also reported.7 In this paper, we describe synthesis and biological evaluation for a series * To whom correspondence should be addressed. Phone: (858) 7944860. Fax: (858) 794-4931. E-mail:
[email protected]. † Department of Organic Chemistry, Chemical Diversity Research Institute. ‡ ChemDiv, Inc. § Department of Medicinal Chemistry, Chemical Diversity Research Institute. # Department of Molecular Biology and HTS, Chemical Diversity Research Institute.
of novel nonpeptide small-molecule inhibitors of caspase-3 having general formula I.
The synthesis of the 4-substituted pyrrolo[3,4-c]quinoline-1,3-dione ring system was accomplished according to a sequence of reactions shown in Scheme 1. Commercially available chloride 1 was reacted with morpholine in 1,4-dioxane to give sulfonyl amide 2. The latter was refluxed in a AcOH/water (1:1 v/v) mixture to afford 8-sulfamoyl isatin 3. Dicarboxylic acids 5 were prepared via a Pfitzinger reaction9 of 3 with keto esters 4a,b under strong alkali conditions. Acids 5 were then converted into furandiones 6a,b upon the reaction with acetic anhydride. Reactions of anhydrides 6a,b with 2-aminoethanol smoothly led to imides 7a,b. Scheme 1a
a Reagents and conditions: (a) morpholine, 1,4-dioxane, room temp, 1 h, 92%; (b) AcOH/H2O (1:1), reflux, 12 h, 89%; (c) KOH in H2O, room temp, 12 h, 60-87%; (d) Ac2O, 100 °C, 3 h, 80-88%; (e) 2-aminoethanol, pyridine, room temp, 1 h, then Ac2O, 80 °C, 30 min, 60-65%.
The second group of 4-substituted pyrrolo[3,4-c]quinoline-1,3-diones was obtained utilizing the commercially available 2,3-dioxo-2,3-dihydro-1H-indole-5-sulfonate 8 as starting reactant. According to this approach depicted in Scheme 2, 8 was treated with keto esters 9a,b under the conditions of Pfitzinger reaction to afford the corresponding dicarboxylic acids 10a,b. Acids 10a,b were converted into furandiones 11a,b upon reaction with acetic anhydride in dry pyridine. Compounds 11a,b were successively treated with 2-aminoethanol and acetic anhydride in pyridine to afford the corresponding imides 12a,b. Sulfonyl chlorides 13a,b were then synthesized from 12a,b by using POCl3 in tetramethylene
10.1021/jm048987t CCC: $30.25 © 2005 American Chemical Society Published on Web 04/29/2005
Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3681
Scheme 2a
Scheme 4a
a Reagents and conditions: (a) CH (CN) , N-methylmorpholine, 2 2 MeOH, reflux, 30 min, 96%; (b) concentrated HCl/AcOH (1:1), reflux, 12 h, 43%; (c) Ac2O, 100 °C, 30 min, 91%; (d) 2-aminoethanol, pyridine, 80 °C, 15 min, then Ac2O, 80 °C, 1 h, 86%; (e) POCl3, reflux, 1 h, 96%.
Scheme 5a a Reagents and conditions: (a) LiOH in H O, room temp, 12 h, 2 60-85%; (b) Ac2O, pyridine, 100 °C, 30 min, 80-88%; (c) 2-aminoethanol, pyridine, room temp, 1 h, then Ac2O, 80 °C, 30 min, 75-97%; (d) POCl3, sulfolane, 80 °C, 2 h, 65-85%; (e) morpholine, 1,4-dioxane, 60 °C, 30 min, 65-95%.
Scheme 3a
a Reagents and conditions: (a) 4-(trifluoromethyl)benzaldehyde, ZnCl2, Ac2O, 110 °C, 70 h, 20%.
sulfone (sulfolane). The reaction proceeded at 80 °C and smoothly afforded the desired product. Finally, morpholides 14a,b were obtained from the reaction of 13a,b with morpholine in 1,4-dioxane at 60 °C. Compound 15 was obtained according to a modification of the reported procedure10 by condensation of 4-methyl derivative 7a with 4-(trifluoromethyl)benzaldehyde in acetic anhydride in the presence of ZnCl2 (Scheme 3). Scheme 4 shows the synthesis of 4-chloride intermediate 20, which was used for the synthesis of 4S- and 4N-substituted pyrrolo[3,4-c]quinoline-1,3-diones. Condensation of 3 with malononitrile led to intermediate 16, which was converted into dicarboxylic acid 17 by reaction with a mixture of concentrated HCl and AcOH. Acid 17 was converted into furandione 18 upon reaction with acetic anhydride. Compound 18 was treated with 2-aminoethanol and acetic anhydride in pyridine at 80 °C, and the resulting imide 19 was reacted with POCl3 to afford the desired chloride 20. Chloride 20 easily reacted with nucleophilic agents (Scheme 5) such as thiourea, methyl mercaptoacetate, morpholine, diethyl (2R)-2-amino-2-methylsuccinate, and 3-chloro-4-fluoroaniline to smoothly afford the corresponding 4S- and 4N-substituted products 21a,b and 22a-c. Compound 21a was prone to rapid spontaneous hydrolysis leading to the corresponding thiol; the latter
a Reagents and conditions: (a) (for 21a) (NH ) CS, i-PrOH, 2 2 reflux, 30 min, 75%; (b) (for 21b) HSCH2CO2Me, dimethoxyethane, Cs2CO3, ultrasonic bath, room temp, 1 h, 65%; (c) morpholine (for 2a) or diethyl (2R)-2-amino-2-methylsuccinate (for 22b) or 3-chloro4-fluoroaniline (for 22c), i-PrOH, reflux, 20-60 min, 45-77%; (d) m-CPBA, CH2Cl2, 0 °C, 1 h, 70%; (e) m-CPBA, CH2Cl2, room temp, 4 h, then 40 °C, 1.5 h, 56%.
was also unstable and underwent slow oxidation, which led to a disulfide dimer. Oxidation of sulfide 21b with m-chloroperbenzoic acid (m-CPBA) in dichloromethane at 0 °C afforded sulfinate 23. When the same reaction was performed at higher temperature (∼40 °C), sulfonate 24 was formed. Finally, Suzuki coupling11 of 20 with 3-chloro-4fluorophenylboronic acid, 4-(N,N-dimethylaminocarbonyl)phenylboronic acid, or 3-pyridylboronic acid in the presence of PdCl2(PPh3)2 afforded the corresponding 4-aryl substituted compounds 25a-c (Scheme 6). Compounds 7a,b, 14a,b, 15, and 19-25 have been tested for their ability to inhibit caspase-3 catalyzed proteolytic breakdown of its fluorogenic substrate, AcDEVD-AMC. The synthesized 1,3-dioxo-2,3-dihydro-1Hpyrrolo[3,4-c]quinolines of general formula I displayed high activity in this in vitro caspase-3 inhibition assay (Table 1). The most active compounds within this series have alkyl, aryl, and heteroaryl 4-substituents. They demonstrated inhibitory activity (IC50) in the 20-60 nM range. 4-Hydroxy- and 4-chloro-substituted derivatives (19 and 20) and 4-sulfanyl derivatives with their oxidized analogues (21a,b, 23, and 24) are significantly
3682
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Letters
Scheme 6a
a Reagents and conditions: (a) 3-chloro-4-fluorophenylboronic acid (for 25a) or 4-[(dimethylamino)carbonyl]phenylboronic acid (for 25b) or 3-pyridylboronic acid (for 25c), dimethoxyethane, PdCl2(PPh3)2, Na2CO3, H2O, reflux, 4 h, 19-25%.
Figure 1. Caspase-3 inhibition curves in the presence of different concentrations of the fluorogenic substrate.
Table 1. In Vitro Caspase-3 Inhibitory Potency of 4-Substituted 2-(2-Acetyloxyethyl)-8-(morpholine-4sulfonyl)pyrrolo[3,4-c]quinoline-1,3-diones of General Formula I compd
R
IC50, nM
7a 7b 14a 14b 15 19 20 21a 21b 22a 22b 22c 23 24 25a 25b 25c
Me i-Pr Ph 2-furyl 4-CF3-C6H4-CHCH OH Cl S-C(NH)NH2 S-CH2CO2Me 1-morpholyl EtOC(O)-CH2-CH(CO2Et)-NH 3-Cl-4-F-C6H3-NH SO-CH2CO2Me SO2-CH2CO2Me 4-(Me2NCO)-C6H4 3-Cl-4-F-C6H3 3-pyridyl
23 ( 2 40 ( 3 27 ( 3 36 ( 4 33 ( 3 10900 ( 3800 294 ( 16 255 ( 20 1290 ( 30 15350 ( 3500 5190 ( 2700 27900 ( 3300 352 ( 21 362 ( 26 58 ( 6 33 ( 4 33 ( 5
less potent (IC50 ) 250-1300 nM). The less active compounds within the synthesized set have alkyl- and arylamino 4-substituents (IC50 ) 5-28 µM). It is suggested that electrophilicity of imide carbonyls, which can be affected by different types of 4-substituents, plays an essential role in the activity of the studied compounds. In accordance with this suggestion, we have observed a clear correlation between the inhibitory activity and nucleophilicity of the 4-substituents (see Supporting Information). The mechanism of inhibition has been assessed for 7a, which is the most active in this series. The noncompetitive character of the inhibition has been demonstrated in the experiments, in which the inhibitory potency was measured in the presence of increasing concentration of the substrate Ac-DEVD-AMC (Figure 1). The incremental increase in the substrate concentration did not shift the inhibition curves to the right as one would expect for competitive inhibition. An IC50 of 23 ( 6 nM was found for 7a from these curves. Parallel experiments demonstrated that the IC50 value for AcDEVD-CHO, a potent tetrapeptide inhibitor of caspase3, was equal to 3.1 ( 0.2 nM under the same experimental conditions. Inhibition by 7a was reversible, as demonstrated by an experiment in which a 10-fold concentration of the compound was preincubated for 1 h with 10-fold caspase-3 concentration followed by 10fold dilution of the mixture into a standard assay buffer to a final compound concentration of 30 nM. At 1.5 h
Figure 2. Studies on mechanism of recombinant human caspase-3 inhibition by 7a.
after dilution, the measured activity of the sample was practically identical to that of inhibition in control samples, which were preincubated at normal concentrations of the compound and enzyme. The plots also demonstrate the noncompetitive character of inhibition because the inhibitor decreases Vmax without effecting the apparent Km (Figure 2). The same conclusion can be made from an analysis of Lineweaver-Burk plots (see Supporting Information). The dissociation constant Km for Ac-DEVD-AMC calculated from these plots was estimated to be 6 ( 2 µM at 95% confidence intervals. The selectivity of 7a was estimated in the reactions with a panel of active recombinant human caspases-1-9 and the specific substrates for each individual enzyme. Compound 7a was the most potent against caspase-3, but the level of selectivity versus the remaining caspases was modest. To assess the antiapoptotic activity of 7a in a cellbased assay, we measured the viability of human Jurkat T cells treated with 10 µM staurosporin. As a positive control, we used a known cell-permeable apoptosis inhibitor Z-VAD-FMK. Compound 7a demonstrated a higher level of protection than the peptide inhibitor, as can be seen from the cell viability values (Figure 3). These data also suggest, though indirectly, good cell permeability of 7a. In summary, we synthesized a series of novel nonpeptide caspase-3 inhibitors based on a 1,3-dioxo-2,3dihydro-1H-pyrrolo[3,4-c]quinoline molecular scaffold. The inhibitory activity depends on the nature of the substituent in position 4 of this heterocyclic system. Compounds 7a and 14a with 4-methyl and 4-phenyl substituents are the most potent compounds (IC50 ) 23 and 27 nM, respectively). Our primary data suggest noncompetitive and reversible character of caspase-3
Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3683
(2)
(3)
(4)
(5)
Figure 3. Protective effect of 7a in a model of staurosporininduced apoptosis in human Jurkat T cells.
inhibition. The selected compound 7a showed moderate selectivity against caspase-3 and marked antiapoptotic activity in a cell-based assay. Further SAR exploration studies are continuing. Acknowledgment. The authors thank Caroline T. Williams (Department of Analytical Chemistry, ChemDiv, Inc.) for NMR and LCMS data. The authors also thank the colleagues of the Scripps Center for Mass Spectrometry (La Jolla, CA) for HRMS data. Supporting Information Available: Synthesis procedures and analytical data for intermediates and final products (1H NMR, 13C NMR, HRMS), experimental procedures for the biological tests, inhibition kinetics, and selectivity data for 7a, and data on the effect of 4-substituents on the caspase-3 inhibitory activity. This material is available free of charge via the Internet at http://pubs.acs.org.
References (1) (a) Denault, J.-B.; Salvesen, G. S. Caspases: keys in the ignition of cell death. Chem. Rev. 2002, 102, 4489-4499. (b) Stennicke, H. R.; Ryan, C. A.; Salvesen, G. S. Reprieval from execution: the molecular basis of caspase inhibition. Trends Biochem. Sci. 2002, 27, 94-101. (c) O’Brien, T.; Lee, D. Prospects for caspase inhibitors. Mini-Rev. Med. Chem. 2004, 4, 153-165. (d) Earn-
(6)
(7) (8)
(9)
(10) (11)
shaw, W. C.; Martins, L. M.; Kaufmann, S. H. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem. 1999, 68, 383-424. (a) Porter, A. G.; Janicke, R. U. Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 1999, 6, 99-104. (b) Nuttall, M. E.; Lee, D.; McLaughlin, B.; Erhardt, J. A. Selective inhibitors of apoptotic caspases: implications for novel therapeutic strategies. Drug Discovery Today 2001, 6, 85-91. Chapman, J.; Magee, W.; Stukenbrok, H.; Beckius, G.; Milici, A.; Tracey, W. A novel nonpeptidic caspase-3/7 inhibitor, (S)(+)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin reduces myocardial ischemic injury. Eur. J. Pharmacol. 2002, 456, 5968. Scott, C. W.; Sobotka-Briner, C.; Wilkins, D. E.; Jacobs, R. T.; Folmer, J. J.; Frazee, W. J.; Bhat, R. V.; Ghanekar, S. V.; Aharony, D. Novel small molecule inhibitors of caspase-3 block cellular and biochemical features of apoptosis. J. Pharmacol. Exp. Ther. 2003, 304, 433-440. Lee, D.; Long, S. A.; Murray, J. H.; Adams, J. L.; Nuttall, M. E.; Nadeau, D. P.; Kikly, K.; Winkler, J. D.; Sung, C. M.; Ryan, M. D.; Levy, M. A.; Keller, P. M.; DeWolf, W. E., Jr. Potent and selective nonpeptide inhibitors of caspases 3 and 7. J. Med. Chem. 2001, 44, 2015-2026. Ganjian, I.; Khorshidi, M.; Lalezari, I. Synthesis and cytotoxic activity of 2-dialkylaminoalkyl-1,3-dihydropyrrolo[3,4-c]quinoline-1,3-diones and 6-(2-dimethylaminoethyl)-1H-dibenz[c,e]azepine-5,7-dione. J. Heterocycl. Chem. 1991, 28, 1173-1175. Shaw, K.; Yuan, J. Certain pyrroloquinolinones; a new class of GABA brain receptor ligands. U.S. Patent 5,604,235, 1994; Chem. Abst. 1994, 120, 106976j. (a) Ivachtchenko, A. V.; Kobak, V. V.; Il’yin, A. P.; Trifilenkov, A. S.; Busel, A. A. New scaffolds for combinatorial synthesis. II. 6-Sulfamoylquinolinecarboxylic acids. J. Comb. Chem. 2003, 5, 645-652. (b) Ivachtchenko, A.; Khvat, A.; Kysil, V.; Maliartchuk, S.; Tkachenko, S.; Okun, I. New potent heterocyclic caspase-3 and apoptosis inhibitor. Drugs Future 2004, 29 (Suppl. A), 191. (c) Ivachtchenko, A. V.; Kobak, V. V.; Kisel, V. M.; Kuzovkova, Y. A.; Il’in, A. P.; Kravchenko, D. V.; Tkachenko, S. E.; Khvat, A. V.; Okun, I. M. Quinoline-carboxylic acids, their derivatives, targeted library, pharmaceutical compositions and methods of their preparation and use. WO 2004/078731, 2004; PCT RU 2004/000081, 2004. (a) Manske, R. H. The chemistry of quinolines. Chem. Rev. 1942, 30, 113-144. (b) Bergstrom, F. W. Heterocyclic nitrogen compounds. Part IIA. Hexacyclic compounds: pyridine, quinoline, and isoquinoline. Chem. Rev. 1944, 35, 77-277. Campaigne, E.; Hutchinson, J. H. Some N-substituted 2-aminoand 2-methylquinoline-3,4-dicarboximides. J. Heterocycl. Chem. 1970, 7, 655-659. Miyaura, N.; Suzuki, A. Palladium-catalyzed cross-coupling reactions of organoboron compounds. Chem. Rev. 1995, 95, 2457-2483.
JM048987T
